Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0034—Urogenital system, e.g. vagina, uterus, cervix, penis, scrotum, urethra, bladder; Personal lubricants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
  • A61P5/30—Oestrogens

Definitions

• the present invention relates to a method of increasing libido in a woman, said method comprising administering an effective amount of an estrogenic component to said woman
• libido defined as the urge to engage in sexual activity and intercourse
• libido is an important component of an individual's well-being.
• Low or decreased libido is a common complaint in women. Such complaints are observed in pre-, peri- as well as post- menopausal women.
• a low libido is characterised by a lack of interest in sexual intercourse and/or the lack of ability to achieve orgasm.
• a decreased libido may be accompanied by a decrease in intensity of orgasm. It is important to note that a decrease in libido is often associated with a profound sense of loss of a once normal and active interest in sexual activity.
• US 6,284,263 (Place) is concerned with a method of treating sexual dysfunction in female individuals, comprising bucally administering a therapeutically effective amount of an androgenic agent, a progestin and an estrogen.
• the US-patent specifically mentions the following estrogens: 17 ⁇ -estradiol, 17 ⁇ -estradiol, ethinyl estradiol, pharmaceutically acceptable esters and ethers of 17 ⁇ -estradiol, 17 ⁇ -estradiol and ethinyl estradiol, estriol, estriol succinate, polyestrol phosphate, estrone, estrone acetate, estrone sulfate, piperazine estrone sulfate, quinestrol, mestranol and conjugated equine estrogens.
• vaginal atrophy and dyspareunia are a common cause of sexual dysfunction.
• biogenic estrogens i.e. estrogens that occur naturally in the human body
• the half-life is around 1 hour.
• blood serum levels of such biogenic estrogens tend to fluctuate considerably.
• the serum concentration is usually several times higher than the optimum concentration.
• serum concentrations will quickly decrease to a level where the estrogen is no longer physiologically active.
• the most important synthetically altered estrogenic steroid is 17 -ethinyl estradiol (EE). This estrogen is dominant in oral hormonal contraception. Apart from EE, mestranol has been used in a few cases; mestranol is a "prodrug” that is metabolised to EE in the organism.
• the liver is a target organ for estrogens.
• the secretion activity that is affected by estrogens in the human liver includes increased synthesis of transport proteins CBG, SHBG, TBG, several factors that are important for the physiology of blood clotting, and lipoproteins.
• DES diethylstilbestrol
• Other undesirable side-effects that have been reported in relation to the use of synthetic estrogens include fluid retention, nausea, bloating, chlolelithiasis, headache, breast pain and an enhanced risk of breast cancer with longer term usage.
• the inventors have unexpectedly found that a special group of estrogenic substances do not exhibit the aforementioned drawbacks and can be used very effectively to improve libido in women.
• These estrogenic substances are represented by the following formula
• R ls R 2 , R 3 , R 4 independently are a hydrogen atom, a hydroxyl group or an alkoxy group with 1-5 carbon atoms; each of R 5 , R 6, R 7 is a hydroxyl group; no more than 3 of Ri, R 2 , R 3 , are hydrogen atoms.
• These estrogens are different from the estrogens commonly applied in estrogen replacement therapy, i.e. ethinyl estradiol, estradiol and its esters such as the acetate, valerate or benzoate, mestranol, the conjugated equine estrogens and estrone sulfate.
• estradiol is 1,3,5 (10)-estratrien-3, 15 ,16 ,17 ⁇ -tetrol, also known by the names of estetrol, oestetrol and 15 ⁇ -hydroxyestriol.
• Estetrol is an estrogen that is produced by the fetal liver during human pregnancy.
• estetrol is a weak estrogen.
• the estrogenic potency of estetrol has been found to be lower than that of another biogenic estrogen, namely, 17 ⁇ -estradiol, which is considered to be a relatively weak estrogen (e.g. compared to ethinyl estradiol).
• US 5,468,736 (Hodgen) describes a method of hormone replacement therapy involving the administration of estrogen together with an amount of antiprogestin (antiprogestogen), which inhibits estrogen-induced endometrial proliferation in women.
• antiprogestogen antiprogestogen
• Example 3 the combined use of estetrol and lilopristone is mentioned. No clues are given in the examples as to the mode and frequency of administration or regarding the dosage level employed.
• a disadvantage associated with the use of antiprogestogens, such as lilopristone is the risk of inducing abnormal endometrial morphology, i.e. cystic hyperplasia, as has been observed in women who received an antiprogestogen treatment against endometriosis (Murphy et al., 1995. Fertil.
• antiprogestogens are a well-known abortive agent and consequently should not be used by women at a fertile age who wish to conceive.
• the benefits of the present invention may be realised without the application of an antiprogestogen.
• estetrol and estetrol-like substances have relatively low estrogenic potency, they may effectively be employed in the present method because their low potency is compensated for by a relatively high metabolic stability, as demonstrated by a long half-life.
• estetrol-like substances may be explained by the relatively high affinity for the estrogen receptor ⁇ (ER ⁇ ) as compared to the estrogen receptor ⁇ (ER ⁇ ).
• ER ⁇ estrogen receptor ⁇
• the latter characteristic is an unique feature of the estrogenic substances employed in the present method.
• the relatively high affinity of the present estrogenic substances for the ER ⁇ receptor, or conversely the relatively low affinity for the ER ⁇ receptor is believed to be somehow associated with the high efficacy of the present substances as libido enhancers.
• Another advantageous property of the present estrogenic substances resides in the fact that sex hormone-binding globulin (SHBG) hardly binds these estrogenic substances, meaning that, in contrast to most known estrogens, serum levels are representative for bio- activity and independent of SHBG levels.
• SHBG sex hormone-binding globulin
• estrogenic substances are derived from their relative insensitivity to interactions with other drugs (drug-drug interactions). It is well known that certain drugs may decrease the effectiveness of estrogens, such as ethinyl estradiol, and other drugs may enhance their activity, resulting in possibly increased side-effects. Similarly estrogens may interfere with the metabolism of other drugs. In general, the effect of other drugs on estrogens is due to interference with the absorption, metabolism or excretion of these estrogens, whereas the effect of estrogens on other drugs is due to competition for metabolic pathways.
• estrogen-drug interactions occurs with drugs that may induce hepatic microsomal enzymes which may decrease estrogen plasma levels below therapeutic level (for example, anticonvulsant agents; phenytoin, primidone, barbiturates, carbamazepine, ethosuximide, and methosuximide; antituberculous drugs such as rifampin; antifungal drugs such as griseofulvin).
• drugs that may induce hepatic microsomal enzymes which may decrease estrogen plasma levels below therapeutic level for example, anticonvulsant agents; phenytoin, primidone, barbiturates, carbamazepine, ethosuximide, and methosuximide; antituberculous drugs such as rifampin; antifungal drugs such as griseofulvin.
• the present estrogenic substances are less dependent on up- and downregulation of microsomal liver enzymes (e.g. P450's) and also are less sensitive to competition with other P450 substrates. Similarly, they do not interfere significantly in
• the above observations serve to explain why the estrogenic substances of the invention can advantageously be used in a method of increasing libido in women.
• the present estrogenic substances exhibit a surprisingly long in vivo half-life in combination with a relatively high affinitiy for the ER ⁇ receptor which is believed to play a crucial role in sexual behaviour.
• the present estrogenic substances hardly suffer from drug-drug interactions and thus produce a very consistent, i.e. predictable, impact.
• the efficacy of the estrogenic substances of the invention is highly reliable.
• the present invention is specifically concerned with a method of increasing libido in a woman which comprises administering to said woman an effective amount of an estrogenic component selected from the group consisting of: substances represented by the following formula
• Ri , R 2 , R 3 , R* independently are a hydrogen atom, a hydroxyl group or an alkoxy group with 1-5 carbon atoms; each of R 5 , R j R 7 is a hydroxyl group; no more than 3 of Ri , R 2 , R 3 , j are hydrogen atoms; precursors capable of liberating a substance according to the aforementioned formula when used in the present method; and mixtures of one or more of the aforementioned substances and/or precursors.
• estrogenic component encompass substances that are capable of triggering an estrogenic response in vivo, as well as precursors that are capable of liberating such an estrogenic component in vivo when used in accordance with the present invention.
• estrogenic components In order for estrogenic components to trigger such a response they normally have to bind to an estrogen receptor, which receptors are found in various tissues within the mammalian body.
• the present invention not only encompasses the use of estrogenic components specifically mentioned in this application, but also metabolites of these hormones that display comparable in vivo functionality. In this context it is observed that, for instance, estriol is a metabolite of 17beta-estradiol.
• estrogenic substances as used in this document does not encompass tritium ( 3 H) labeled estrogenic substances such as tritium labeled estetrol.
• the present estrogenic substances are distinct f om both the biogenic and synthetic estrogens that are commonly applied in pharmaceutical formulations in that they contain at least 4 hydroxyl groups.
• the present substances are special in that the 5 membered ring in the steroid skeleton comprises 3 hydroxyl substituents rather than 0-2.
• Known estrogens that contain at least 4-hydroxyl groups and derivatives thereof are:
• the estrogenic substance applied as the active component in the present composition is a so called biogenic estrogen, i.e. an estrogen that occurs naturally in the human body, a precursor of a biogenic estrogen or mixtures thereof. Because biogenic estrogens are naturally present in the fetal and female body, side-effects are not expected to occur, particularly not if the serum levels resulting from the exogenous administration of such estrogens do not substantially exceed naturally occurring concentrations.
• estetrol serum levels in the fetus are several times higher than those found in pregnant females and knowing that the fetus is particularly vulnerable, estetrol is deemed to be a particularly safe biogenic estrogen. Side-effects are not expected to occur, particularly not if the serum levels resulting from the exogenous administration of such estrogens do not substantially exceed naturally occurring (fetal) concentrations.
• synthetic estrogens such as ethinyl estradiol there is a
• the estrogenic substance contains 4 hydroxyl groups.
• R ! preferably represents a hydrogen atom.
• at least 2 more preferably at least 3 of the groups Ri, R 2 , R 3 and -j represent a hydrogen atom.
• the estrogenic substances according to the formula encompass various enantiomers since the carbon atoms that carry hydroxyl-substituents R 5 , R 6 and R are chirally active.
• the present estrogenic substance is 15 ⁇ -hydroxy substituted.
• the substance is 16 ⁇ -hydroxy substituted.
• the substances is 17 ⁇ -hydroxy substituted.
• the estrogenic substances are 15 ⁇ ,16 ,17 ⁇ -trihydroxy substituted.
• R 3 represents a hydroxyl group or an alkoxy group.
• the groups R ls R 2 and R 4 represent hydrogen atoms, in which case, if R 3 , R 5 , R 6 and R 7 are hydroxyl groups, the substance is 1,3,5 (10)-estratrien-3, 15,16,17-tetrol.
• a preferred iso er of the latter substance is 1,3,5 (10)-estratrien-3, 15 ⁇ ,16 ,17 ⁇ -tetrol (estetrol).
• the invention also encompasses the use of precursors of the estrogenic substances that constitute the active component in the present method. These precursors are capable of liberating the aforementioned estrogenic substances when used in the present method, e.g. as a result of metabolic conversion.
• precursors are preferably selected from the group of androgenic precursors as well as derivatives of the present estrogenic substances.
• Suitable examples of androgenic precursors include androgens that can be converted into the present estrogenic substances through in vivo aromatisation.
• Examples of derivatives of the present estrogenic substances that can suitably be used as precursors include such substances wherein the hydrogen atom of at least one of the hydroxyl groups has been substituted by an acyl radical of a hydrocarbon carboxylic, sulfonic acid or sulfamic acid of 1-25 carbon atoms; tetrahydrofuranyl; tetrahydropyranal; or a straight or branched chain glycosidic residue containing 1-20 glycosidic units per residue.
• Typical examples of precursors which can suitably be used in accordance with the invention are esters that can be obtained by reacting the hydroxyl groups of the estrogenic substances with substances that contain one or more carboxy (M* OOC-) groups, wherein M* represents a hydrogen or (akali)metal cation.
• the precursors are derivatives of the estrogenic substances, wherein the hydrogen atom of at least one of the hydroxyl groups in said formula has been substituted by -CO-R, wherein R is a hydrocarbon radical comprising from 1-25 carbon atoms.
• R is hydrogen, or an alkyl, alkenyl or aryl radical comprising from 1-20 carbon atoms.
• the present method encompasses protocols wherein the estrogenic component is administered at predetermined, regular intervals as well as a protocol wherein the estrogenic component is administered on demand at the moment a woman wishes to increase her libido.
• the term "libido" has been defined above as the urge to engage in sexual activity and intercourse. It is noted that such an urge may be affected by both psychological and fysiological factors (e.g. vaginal atrophy and dyspareunia).
• the present method is employed to increase the libido of a female who does not suffer from decreased libido as a result of vaginal atrophy and/or dyspareunia.
• the present method may suitably employ enteral or parenteral administration of the estrogenic component.
• parenteral administration as used in here encompasses transdermal, intranasal, intravaginal, pulmonary, buccal, subcutaneous, intramuscular and intra-uterine administration.
• enteral administration includes oral as well as rectal administration.
• the mode of administration is selected from the group consisting of oral, transdermal, intranasal, intravaginal, pulmonary, rectal, buccal, subcutaneous, intramuscular or intra-uterine administration. More preferably the mode of administration is selected from the group consisting of oral, transdermal, subcutaneous, intramuscular, intranasal, pulmonary and vaginal administration.
• the present method employs oral, intranasal, intravaginal or rectal administration. Even more preferably the present method employs oral or intranasal administration.
• intranasal, rectal, buccal and pulmonary administration are ideally suited for (at least) once daily administration.
• Transdermal admimstration is advantageously applied at frequencies between once a day and once a month.
• Intravaginal and intra-uterine administrations are advantageously operated at administration frequencies between once weekly and once monthly.
• Subcutaneous and intramuscular administration are suitably done in the form of depot injections at intervals of 1 week to 6 months, preferably at intervals of 4 weeks to 3 months.
• the present method preferably utilises administration intervals of 1 day, 1 week or 1 month.
• Administration intervals of 1 day, 1 week or 1 month.
• Regimens that employ once daily oral or intranasal administration, once weekly transdermal or once monthly intravaginal or subcutaneous administration are particularly preferred.
• the estrogenic component is preferably administered in an amount effective to achieve a blood serum concentration of at least 1 nanogram per litre, more preferably of at least 10 nanogram per litre, most preferably at least 100 nanogram per litre.
• the resulting blood serum concentration of the estrogenic component will not exceed 100 ⁇ g per litre, preferably it will not exceed 50 ⁇ g per litre, more preferably it will not exceed 25 ⁇ g per litre.
• the estrogenic component is usually administered in an amount of less than 1 mg per kg of bodyweight per day, preferably of less than 0.4 mg per kg of bodyweight per day.
• the administered amount is at least 5 ⁇ g per kg of bodyweight per day.
• Oral administration of the active component is preferably done in an amount of less than 400 ⁇ g per kg of bodyweight per day, preferably of less than 200 ⁇ g per kg of bodyweight per day.
• the orally administered amount is at least 5 ⁇ g per kg of bodyweight per day.
• the present method comprises administering to a woman in need of enhanced libido an effective amount of the present estrogenic component.
• the amounts needed to be effective will differ from individual to individual and are detemiined by factors such as the individual's endogenous estrogen levels, body weight, route of administration and the efficacy of the particular estrogen substance used.
• the estrogenic component is administered in an dosage of at least 0.05 mg per day, preferably of at least 0.1 mg per day.
• the maximum dosage normally does not exceed 40 mg per day, preferably it does no exceed 20 mg per day.
• the present method is particularly suited for increasing libido in hypoestrogenic women.
• a typical example of hypoestrogenic women are menopausal and postmenopausal females. It is noted that many menopausal and postmenopausal women undergo estrogen replacement therapy, which usually involves the combined administration of an estrogen (often 17- ⁇ estradiol) and a progestogen. Since 17- ⁇ estradiol is deemed to be less effective in restoring libido than the present estrogenic substances, it is deemed advantageous to administer these estrogenic substances, in addition to or instead of 17- ⁇ estradiol, to improve the libido of menopausal or postmenopausal women who are using estrogen replacement therapy.
• the method employs oral administration of the active estrogenic component.
• oral administration as used in here also encompasses oral gavage administration.
• estetrol and related estrogenic substances may advantageously be administered orally.
• estetrol-like substances results from the combination of special pharmacokinetic and pharmacodynamic properties of these substances.
• estetrol-like substances are surprisingly high and that their in vivo half-life is considerably longer than that of biogenic estrogens. Thus, even though estetrol and estetrol-like substances have relatively low estrogenic potency, they may effectively be administered orally.
• estetrol and estetrol-like substances Another important advantage of oral administration of estetrol and estetrol-like substances resides in the fact that the hepatic effects of these substances are deemed to be minimal since they are hardly metabolised during the so called "first pass".
• the first-pass effect of drugs given orally refers to the process of drug degradation by the liver during a drug's transition from initial ingestion to circulation in the blood stream. After resorption from the intestinal lumen, orally applied active ingredients enter the organism via the liver. This fact is of specific importance for estrogenic agents as the liver is a target organ for estrogens; oral intake of estrogens results in strong estrogenic effects in the liver.
• biogenic estrogens when applied orally, result in clear responses of hepatic parameters, such as increase of SHBG, CBG, angiotensinogen and HDL (high density lipoprotein). These hepatic effects of estrogens are also observed when equine estrogen formulations (so-called conjugated estrogens) are used. Ethinyl estradiol and diethylstilbestrol (DES) have an even greater hepatic estrogenicity.
• a progestogenic component to inhibit estrogen stimulation of the endometrium (Beral et al., 1999. J. Epidemiol. Biostat., 4, 191-210) or to administer a progestogenic component at least during a period often days at least every three months.
• the progestogen used in the present method is selected from the group consisting of progesterone, desogestrel, etonogestrel, gestodene, dienogest, levonorgestrel, norgestimate, norethisterone, drospirenone, trimegestone, dydrogesterone, precursors of these progestogens and mixtures thereof.
• the progestogenic component is suitably administered in an amount which is equivalent to an oral dosage of 30-750 ⁇ g levonorgestrel, more preferably of 50-400 ⁇ g levonorgestrel.
• the present method employs the co- administration of the estrogenic component with an androgenic component. It was found that in some cases the combined administration of an estrogenic and an androgenic component is more effective than the administration of the estrogenic componentj_er._e in achieving improved libido.
• the androgenic component may suitably be selected from the group consisting of dehydroepiandrosterone (DHEA); DHEA-sulphate; testosterone; testosterone esters such as testosterone undecanoate , testosterone propionate, testosterone phenylpropionate, testosterone isohexanoate, testosterone enantate, testosterone bucanate, testosterone decanoate, testosterone buciclate; danazol; gestrinone; methyltestosterone; mesterolon; stanozolol; androstenedione; dihydrotestosterone; androstanediol; metenolon; fluoxymesterone; oxymesterone; methandrostenolol; MENT; precursors capable of liberating these androgens when used in the present method and mixtures thereof.
• DHEA dehydroepiandrosterone
• DHEA-sulphate testosterone
• testosterone esters such as testosterone undecanoate , testosterone propionate, testosterone phenylpropionate
• the androgenic component is selected from the group consisting of DHEA, testosterone esters, androstenedione, precursors capable of liberating these androgens when used in the present method and mixtures thereof.
• the androgenic component is selected from the group consisting of dehydroepiandrosterone, esters of testosterone and mixtures thereof.
• the testosterone esters employed in the present method comprise an acyl group which comprises at least 6, more preferably from 8-20 and preferably 9-13 carbon atoms.
• the androgens used in the present method are DHEA and/or testosterone undecanoate. These androgens offer the advantage that they can effectively be used in oral dosage units.
• DHEA testosterone undecanoate and androstenedione are precursors of testosterone and that said precursors per se exhibit virtually no affinity for androgen receptors in the female body.
• the effectiveness of the androgens within the method of the invention is determined by their functionally active form, which may well be different from the form in which they are administered.
• the androgen is preferably provided in an amount equivalent to an oral dosage of at least 5 mg DHEA, which is equivalent to an oral dosage of at least 1 mg testosterone undecanoate. More preferably the androgen is provided in an amount which is equivalent to an oral dosage of a least 10 mg DHEA, most preferably of at least 20 mg DHEA. Usually the androgen dosage employed will not exceed the equivalent of an oral dosage of 250 mg DHEA, which is equivalent to an oral dosage of 50 mg testosterone undecanoate. Preferably the androgen is administered in a dosage which does not exceed the equivalent of an oral dosage of 120 mg DHEA, more preferably it does not exceed the equivalent of an oral dosage 60 mg DHEA.
• Vaginal cornification was chosen as a tissue-specific and estrogen-sensitive endpoint to determine the estrogenicity of estetrol (E4), after both oral and subcutaneous administration, in hypoestrogenic rats.
• E4 estetrol
• 17 ⁇ -ethinylestradiol (EE), 17 ⁇ -estradiol (E2) and vehicle (10% ethanol/sesame oil) served as controls in these bioassays.
• Uterine weight increase in the rat is more commonly used as a measure of estrogenicity.
• uterine weight also responds to progesterone, testosterone, and other agents not characteristically regarded as estrogens.
• follicular fluid from the pig ovary contained a factor(s) that caused cormfication/keratinization of the vaginal epithelium in the rat (Allen and Doisy, 1923, JAMA, 81, 819-821; Allen and Doisy, 1924, Am. J. Physiol, 69, 577-588).
• the so-called vaginal cornification response in rats subsequently provided a bioassay for testing estrogenicity.
• Vaginal epithelial cornification/keratinization in ovariectomized rats can be produced only by compounds considered to be true estrogens (Jones et al, 1973, Fert. Steril. 24, 284-291). Vaginal epithelial cornification/keratinization represents, therefore, a highly selective endpoint to determine the potency of estrogens (Reel et al., 1996, Fund. Appli. Toxicol. 34, 288-305).
• vaginal washings were placed on a glass slide and examined by light microscopy to detect the presence or absence of cornified epithelial cells.
• Vaginal lavages were obtained prior to dosing on days 0-6 and prior to necropsy on day 7.
• the vaginal cornification bioassay was performed in order to determine the estrogenic profile of E4 when given subcutaneously (sc) to ovariectomized adult rats.
• E2 was used as a positive control.
• the vehicle (10% ethanol/sesame oil) served as the negative control.
• Steroids were dissolved in absolute ethanol and then brought to the final concentration with sesame oil (10%o ethanol in sesame oil).
• vaginal estrogenic response occurred in 8/8 rats by day 2 and persisted through day 7 in rats injected sc with 50 ⁇ g/kg/day E2 for 7 days (Table 1).
• Animals treated with the vehicle did not exhibit vaginal epithelial cornification (Table 1).
• the onset of vaginal epithelial cornification was dose-dependent in rats injected sc with 0.1, 0.3, 1.0, and 3.0 mg/kg/day E4 and started at the same day of treatment (Day 2) as observed for E2 (Table 1).
• At 1.0 and 3.0 mg/kg/day E4 all rats showed a vaginal estrogenic response by day 7 (Table 1).
• Table 1 Vaginal estrogenic response in ovariectoi zed rats treated subcutaneously (sc) with 17 ⁇ -estradiol (E2) or estetrol (E4). Data are expressed as the number of rats showing vaginal cornification over the number of rats (ratio) treated.
• vaginal cornification bioassay was performed in order to determine the estrogenic profile of E4 when given orally (po) to ovariectomized adult rats.
• EE was used as a positive control.
• the vehicle (10% ethanol/sesame oil) served as the negative control.
• Steroids were dissolved in absolute ethanol and then brought to the final concentration with sesame oil (10% ethanol in sesame oil).
• vaginal epithelial cornification was observed in all rats (8/8) treated po with either 0.1, 0.3, 1.0, or 3.0 mg/kg/day E4 by day 7 (Table 2), whereas animals treated with the vehicle did not exhibit vaginal epithelial cornification (0/8).
• E4 e.g. 0.1 mg/kg/day
• the onset of vaginal cornification was faster in po-treated (Table 2)than in sc-treated animals (Table 1)), demonstrating estetrol' s superb bioavailability characteristics after oral administration
• Table 2 Vaginal estrogenic response in ovariectomized rats treated orally (po) with 17 ⁇ -ethinyl estradiol (EE) or estetrol (E4). Data are expressed as the number of rats showing vaginal cornification over the number of rats (ratio) treated.
• Rats Female Sprague Dawley rats were equipped with a permanent silatic heart catheter, as described by Kuipers et al. (1985, Gastroenterology, 88, 403-411). Rats were allowed to recover from surgery for 5 days and were than administered 0.05, 0.5, or 5 mg/kg E4 in 0.5 ml arachis oil. For sc administration, E4 was injected in the neck area using a 1 ml syringe and 20g needle. For po administration of E4, rats were lightly anaesthesized with halothene/ N 2 O/O 2 and E4 was directly applied infragastrically using a plastic stomach intubator.
• Plasma samples were subsequently collected via the heart catheter in heparinized tubes at 0.5, 1, 2, 4, 8 and 24 hours. Erythrocytes were removed by centrifugation at 5000xg for 10 minutes at 4°C and blood plasma was stored at -20°C. After thawing the plasma samples, liquid-liquid extraction (hexane/diethylether) was employed to prepare the E4-containing plasma samples for HPLC analysis (Perkin Elmer 200) and tandem mass specfrometry using a PE Sciex 3000 tandem mass spectrometer and APCI interface. With each sample batch, a calibration curve with 6 calibrators was recorded. The calibration curve was calculated using linear regression (correlation coefficient > 0.98), which permitted quantitation of plasma concentrations.
• E4 concentration data were analysed with "WinNonLin, edition 3.1" and involved pharmacokinetic parameters for C max , half-life and AUC 0-24 .
• E4 demonstrated an oral bioavailability equal to the bioavailability obtained with sc administration (80-100 %).
• 5.0 mg/kg E4 absorption kinetics gave rise to an oral bioavailability approximating 30-60% of sc administered E4.
• E4 demonstrated a relatively long half-life of 2-3 hours, enabling the detection of bioactive levels of unconjugated E4 at all time points over a 24 hour interval in the sc and po dosing experiments.
• Example 3 Established competitive steroid binding assays were used to determine the relative binding affinity of estetrol (E4), as compared to 17 ⁇ -ethinylestradiol(EE) and 17 ⁇ -estradiol (E2), to human Estrogen Receptor (ER) ⁇ - and ⁇ -forms.
• Recombinant human ER ⁇ or ER ⁇ proteins were dissolved in binding buffer (10 mM Tris-HCL, pH 7.5, 10% glycerol, 1 mM DTT, 1 mg/ml BSA) and duplicate aliquots were then incubated with [ 3 H]E2 at a final concentration of 0.5 nM, together with a vehicle control (0.4% DMSO), or the same amount of vehicle containing increasing concentrations of unlabeled steroid ligands as competitors. After incubation for 2 h at 25 C, the unbound ligands were removed and the amounts of [ 3 H]E2 bound to either ER ⁇ or ER ⁇ proteins were measured.
• binding buffer 10 mM Tris-HCL, pH 7.5, 10% glycerol, 1 mM DTT, 1 mg/ml BSA
• Biochemical assay results for E4 are presented as the percent inhibition of specific binding in three separate experiments (Table 3).
• Table 3 For comparision of binding affinities of E4, EE and E2 to human ER ⁇ and ER ⁇ proteins, experimentally observed Ki values are shown in Table 4.
• E4 demonstrates a unique binding profile with a strong preference (400%) for binding to the ER ⁇ protein (Table 4).
• Eli values for ER ⁇ protein are more pronounced for EE and E2 steroid ligands (Table 4).
• Human SHBG was purified from transgenic mouse serum, as described previously (Awakumov GV et al., 2000. J Biol Chem 275: 25920-25925).
• the human SHBG prepared in this way was assessed to be >99% pure by polyacrylamide gel electrophoresis under denaturing conditions. Its steroid-binding characteristics are indistinguishable from SHBG in human serum (Awakumov GV et al., 2000. J Biol Chem 275: 25920-25925).
• the in vitro assay involved the use of the purified human SHBG and [ 3 H]DHT or [ 3 H]estradiol as labeled ligands.
• Human SHBG was treated for 30 min at room temperature with a dextran-coated charcoal (DCC) suspension in phosphate buffered saline (PBS) to remove any steroid ligand. After centrifugation (2,000 x g for 10 min) to sediment the DCC, the supernatant containing the human SHBG was diluted in PBS to a concentration of 1 nM based on its steroid binding capacity.
• DCC dextran-coated charcoal
• PBS phosphate buffered saline
• Figure 1 Competitive displacement of [ 3 H]DHT (panel A) and [ 3 H]estradiol (panel B) from the human sex hormone-binding globulin steroid binding site.
• the unlabeled steroid ligands used as competitors were as follows: estetrol (E4), 17 ⁇ -ethinylestradiol (EE2), 17 ⁇ -estradiol (E2), testosterone (T) and 5 ⁇ - dihydrotestosterone (DHT)
• estetrol does not bind at all to human SHBG when tested with either [ 3 H]DHT or [ 3 H]estradiol as labeled ligands.
• Dosage units for oral administration may suitably be processed, together with additives, excipients and/or flavoring agents customary in galenic pharmacy, in accordance with the conventional methods into the usual forms of administration.
• suitable are, in particular, tablets, dragees, capsules, pills, suspensions, or solutions.
• Estetrol tablets 1 ,000 tablets of 185 mg, containing 1.5 mg estetrol, are produced from the following formulation:
• Polyvinylpyrrolidone (Kollidon 25® ex BASF) 13.500 g Lactose 135.795 g Microcrystalline cellulose (Avicel PH 101 ®) 26.250 g
• Anhydrous colloidal silica (Aerosil 200 ®) 1.000 g
• Crospovidone (Polyplasdone XL ®) 4.000 g
• Suitable nontoxic pharmaceutically acceptable carriers for use in a drug delivery system for intranasal administration of the present estogenic component will be apparent to those skilled in the art of nasal pharmaceutical formulations. For those not skilled in the art, reference is made to "Remington's Pharmaceutical Sciences", 4th edition, 1970. Obviously, the choice of suitable carriers will depend on the exact nature of the particular nasal dosage form desired, e.g. whether the estrogenic component is to be formulated into a nasal solution (for use as drops or as a spray), nasal microspheres, a nasal suspension, a nasal ointment or a nasal gel, as well as on the identity of the estrogenic component.
• estetrol 5 mg is combined with 10 mg of Tween 80. That mixture is then combined with a quantity of isotonic saline sufficient to bring the total volume to 50 ml.
• the solution is sterilised by being passed through a 0.2 micron Millipore filter.
• a clinical study is conducted in 40 postmenopausal women who are not using hormone replacement therapy and who do not suffer from vaginal atrophy or dyspareunia.
• coitus frequency and sexual satisfaction are recorded.
• Each participant receives blinded medication in 2 differently labeled bottles, filled with tablets.
• One bottle comprises tablets that contain 2 mg estetrol, whilst the other bottle contains identical placebo tablets.
• the participants are allowed to choose from which bottle to take a tablet, but are instructed not to use more than 1 tablet per 24 hours and not more than 2 tablets a week.
• the total duration of the study is 4 months.
• estetrol containing tablets It is found that during the study participants use significantly more estetrol containing tablets than placebo's. In addition, analysis of the data shows that women who mainly used estetrol containing tablets report improved libido and sexual enjoyment in comparison to baseline. Consequently, it can be concluded that oral administration of 2 mg estetrol leads to an improvement of libido and sexual enjoyment in some of these users, whilst no undesirable side-effects are reported.

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